The quest for therapies able to induce long-term survival of islet allografts in humans remains unfulfilled. New strategies targeting T cell signaling molecules may induce transplant tolerance in animals by qualitatively altering T cell activation. CD45 is a family of transmembrane protein tyrosine phosphatases (PTPases) critically involved in the regulation of T cell receptor-mediated signals. Multiple CD45 isoforms are differentially distributed on subsets of T cells having distinct functions. Antibodies against CD45 can induce tolerance in murine models of islet and renal transplantation. Furthermore, preliminary data demonstrate that a short course of anti- CD45 as sole therapy can induce long-term renal allograft survival in non-human primates. Based on these results, we conclude that CD45 is a potent immunotherapeutic target that clearly has potential in human islet transplantation. However, the mechanism(s) by which anti-CD45 acts are poorly understood, and which human isoforms should ideally be targeted is not yet clear. In vitro evidence suggests that anti-CD45 may induce altered activation signals that have been associated with anergy in a T cell clone. Furthermore, treatment of animals with toleragenic anti-CD45 mAbs induces a shift in CD45 isoform expression towards the lower (CD45RB\Lo)Mr forms. T cells expressing these isoforms preferentially secrete Th2-type cytokines and downregulate autoimmune responsiveness in several animal models. The studies in this proposal are designed to extend our understanding of the mechanisms by which anti-CD45 acts, determine the best isoforms to target, and determine whether the alteration of "signal one" with anti-CD45 is synergistic with agents that block "signal two." In Aim 1, in vitro and in vivo studies will be used to determine the mechanism(s) of action of anti-CD45 at the cellular and biochemical level. Specifically, we will address: (a) whether anti-CD45 induces aberrant signal transduction and whether this results in anergy/altered responsiveness, (b) the role of modified CD45 isoform expression in altered cytokine secretion and function, and (c) whether tolerance is based on the development of anergy, suppression, or both. In Aim 2, we will determine whether anti-CD45 is synergistic with blockade of either the CD40L or CD28 pathways in the generation of tolerance. In Aim 3, we will assess the efficacy of different anti-human CD45 mAbs using an established model of human islet cell transplantation in the Hu-SCID mouse. These studies will provide practical information required before clinical trials in human islet transplantation are initiated, and will also provide novel insight into the induction of tolerance that may allow the development of new therapeutic strategies in the future.